Post-transcription gene silencing occurs when double stranded RNA (dsRNA) is introduced or naturally expressed in cells. RNA interference (RNAi) has been described in plants, nematodes, and Drosophila. This process serves at least two roles. It is used as an innate defense mechanism, and it is also used during development. RNAi may regulate developmental expression of genes via the processing of small, temporally expressed RNAs. Harnessing this ability to respond specifically to dsRNA for target mRNA degradation has been a major advance, allowing for the rapid evaluation of gene function.
Despite the attention given to RNAi research recently, the field is still in the early stages of development. Not all siRNA molecules are capable of targeting the destruction of their complementary RNAs in a cell. As a result, complex sets of rules have been developed for designing RNAi molecules that will be effective. Those having skill in the art expect to test multiple siRNA molecules to find functional compositions. (Ji et al., 2003) Some artisans pool several siRNA preparations together to increase the chance of obtaining silencing in a single study (Ji et al., 2003). Such pools typically contain 20 nM of a mixture of siRNA oligonucleotide duplexes or more (Ji et al., 2003), despite the fact that a siRNA molecule can work at concentrations of 1 nM or less (Holen et al., 2002). This technique can lead to artifacts caused by interactions of the siRNA sequences with other cellular RNAs (“off target effects”) (Scherer et al., 2003). Off target effects can occur when the RNAi oligonucleotides have homology to unintended targets or when the RISC complex incorporates the unintended strand from and RNAi duplex (Scherer et al., 2003). Generally, these effects tend to be more pronounced when higher concentrations of RNAi duplexes are used (Scherer et al., 2003).
In addition, the duration of the effect of an effective RNAi treatment is limited to about four days (Holen et al., 2002). Thus, researchers must carry out siRNA experiments within 2-3 days of transfection with an siRNA duplex or work with plasmid or viral expression vectors to obtain longer term silencing.